Journal: STAR Protocols

Article Title: Protocol for SUM-PAINT spatial proteomic imaging generating neuronal architecture maps in rat hippocampal neurons

doi: 10.1016/j.xpro.2025.103637

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-SV2B (final dilution: 1:200) , Synaptic Systems , Cat#119 111; RRID: AB_11042616.

Techniques: Recombinant, Multiplexing, Electron Microscopy, Modification, Sequencing, Software, Cell Culture, Inverted Microscopy

Journal: bioRxiv

Article Title: Synaptic vesicle glycoprotein 2C (SV2C) modulates dopamine release and is disrupted in Parkinson’s disease

doi: 10.1101/077586

Figure Lengend Snippet: We designed polyclonal SV2C antibodies. (A) hSV2C antibody recognizes SV2C but does not recognize SV2A in transfected HEK293 cells. Pre-incubating the hSV2C antibody with the immunizing peptide (“antigen-blocking”) eliminates antibody reactivity. (B) SV2C is highly colocalized with dopaminergic marker tyrosine hydroxylase (TH) in the substantia nigra pars compacta (SNc) and dorsal striatum (CPu) of mouse. (C) Knocking down endogenous SV2C in Neuro-2a cells (which have no endogenous SV2A or SV2B) using RNAi reduces mSV2C antibody reactivity. (D) Sagittal 3–3’ diaminobenzidine-(DAB) stained sections showing ubiquitous SV2A and SV2B expression compared to SV2C expression enriched in the basal ganglia using the mSV2C antibody. Black arrows indicate areas of highest SV2C staining in the mouse brain: midbrain, dorsal striatum and ventral pallidum. Immunoblots separated by black lines indicate bands that came from the same blot and were cropped for easy comparison between lanes .

Article Snippet: Monoclonal mouse-anti SV2B and mouse- anti synaptotagmin-1 antibodies were purchased from Synaptic Systems.

Techniques: Transfection, Blocking Assay, Marker, Staining, Expressing, Western Blot, Comparison

Journal: bioRxiv

Article Title: Synaptic vesicle glycoprotein 2C (SV2C) modulates dopamine release and is disrupted in Parkinson’s disease

doi: 10.1101/077586

Figure Lengend Snippet: (A) PCR genotyping of SV2C-KO mice generated using the EUCOMM “knockout-first” allele. This construct allowed us to generate several useful lines of mice: a preliminary knockout animal with a cassette creating a frt-flanked gene trap inserted into the second exon (KOF), a line with a floxed exon after breeding KOF animals with a flp-recombinase+ line, and finally SV2C-KO mice following a cross with a nestin-driven Cre-recombinase+ line. Animals used for experiments are denoted by a superscript 1 (WT control) and 2 (SV2C-KO). See methods for primer sequences. (B) SV2C-KO does not result in altered expression of either SV2A or SV2B, nor of dopaminergic markers tyrosine hydroxylase (TH) or dopamine transporter (DAT). (C) Genetic deletion of SV2C ablates mSV2C antibody reactivity in the striatum (CPu) and midbrain. Dotted line delineates dorsolateral striatum where electrochemical recordings were taken. (D-E) SV2C-KO results in a 33% reduction of dopamine content in the dorsal striatum (n=7, p<0. 01) and a 19% reduction in the dopamine metabolite DOPAC.

Article Snippet: Monoclonal mouse-anti SV2B and mouse- anti synaptotagmin-1 antibodies were purchased from Synaptic Systems.

Techniques: Generated, Knock-Out, Construct, Control, Expressing

Journal: Nature

Article Title: Ageing promotes microglial accumulation of slow-degrading synaptic proteins

doi: 10.1038/s41586-025-09987-9

Figure Lengend Snippet: a. In-gel fluorescence images comparing the presence of Alexa 647-clicked and thus BONCAT labeled proteins from cell lysates derived from the indicated cell lines expressing either PheRS* (left) or TyrRS* (right) that either did or did not undergo inhibition of protein synthesis prior to non-canonical amino acid treatment. As shown in the experimental schematic (top), BONCAT-expressing cell lines were treated with protein synthesis inhibitor before being treated with the non-canonical amino acid, which abrogated protein tagging relative to cells not treated with protein synthesis inhibitor. b. In-gel fluorescence image of Alexa 647-clicked and thus BONCAT labeled proteins from total brain lysates derived from young Camk2aCre;MetRS*, Camk2aCre;PheRS*, and Camk2aCre;TyrRS* BONCAT transgenic mice and their respective wildtype background controls. Relative Alexa 647 intensities can be taken as a measure of relative labeling efficiencies. c. Fluorescence images of Alexa 488-clicked and thus BONCAT labeled proteins in cortical tissue sections from young Camk2aCre;PheRS* model. Tissues are co-stained with anti-GFP, which should be co-expressed in all cells expressing PheRS*, and DAPI. d. Fluorescence images of Alexa 594-clicked and thus BONCAT labeled proteins in cortical tissue sections from young Camk2aCre;PheRS* model. Tissues are co-stained with anti-NeuN, a neuronal marker, and DAPI to demonstrate localization of BONCAT labeled proteins to neurons. e. In situ heatmap of Camk2a mRNA expression from the Allen Brain Atlas. f. Schematic of methodology used to enrich BONCAT-labeled proteins from total lysates for LC-MS. g. Overlaid histograms comparing the number of proteins pulled down by the methodology illustrated in (f) in unlabeled (wildtype mice treated with AzF) and labeled samples (Camk2aCre;PheRS* mice treated with AzF). Effect size determined by Cohen’s D. P values determined by two-tailed Kolmogorov-Smirnov Test and two-tailed Wilcoxon Rank Sum Test. h. Histogram showing the number of labeled proteins (from Camk2aCre;PheRS* mice treated with AzF) and their respective percent increase in enrichment over unlabeled proteins (from wildtype mice treated with AzF). i. Box and whisker plots comparing the log 2 -transformed protein intensities for all proteins identified by LC-MS in BONCAT-labeled mice and their respective background controls (wildtype mice treated with the same non-canonical amino acid). Boxes extend from the 25 th to 75 th percentiles and the horizontal line represents the median. Whiskers extend from data minimum to data maximum. n = 2985, 3716, and 2906 unfiltered proteins/replicate for Camk2a;MetRS*, Camk2a;PheRS*, and Camk2a;TyrRS*, respectively. j. Bar chart showing the number of proteins identified without any filtering (black bars), after filtering based on valid values as described in the methods (light grey bars), and after filtering based on valid values, fold change (log 2 (fold change) > 1 over the background) and p value (<0.05, determined from a two-tailed, two-sample students t-test) (light grey bars) for each Camk2aCre;BONCAT line. k. In-gel fluorescence image of Alexa 647-clicked and thus BONCAT labeled proteins of total brain lysates derived young Camk2aCre;PheRS* mice provided 185 mg/kg of azido-phenylalanine (AzF) for a varying number of days (left) and associated quantification of fluorescence intensity of clicked-protein normalized to total protein with the different number of days provided AzF (right). l. In-gel fluorescence image of Alexa 647-clicked and thus BONCAT labeled proteins of total brain lysates derived from young Camk2aCre;PheRS* mice provided varying doses of azido-phenylalanine (AzF) for one week (left) and associated quantification of fluorescence intensity of clicked-protein normalized to total protein with the different doses of AzF provided (right). m. Western blot image of HSP90 and loading control beta-actin on whole brain lysates derived from various BONCAT-labeled models and ages and respective non-labeled controls to show whether BONCAT-labeling induces an HSP90-mediated heat shock response. n. Fluorescence images for microglia (Iba1, green) staining in cortical tissue sections of an AzF-treated Camk2aCre;PheRS*mouse (left), wildtype mouse not administered AzF (middle), and wildtype mouse administered AzF (right) to determine whether BONCAT-labeling induces microgliosis as evaluated by cellular morphology. All sections were additionally clicked with Alkyne 594 and imaged with the appropriate and equal laser settings for Alkyne 594 detection in order to verify the presence of labeled proteins or lack of labeled proteins in each section.

Article Snippet: Primary antibodies were used at the following dilutions: rabbit anti-GFP (Cell Signaling Technology, 2956S) at 1:400; rabbit anti-IBA1 (Fujifilm Wako, 019-19741) at 1:2,000; guinea pig anti-NeuN (Synaptic Systems, 266 004) at 1:1,000; rabbit anti-SATB2 (Synaptic Systems, 327 003) at 1:500; guinea pig anti-parvalbumin (Synaptic Systems, 195 308) at 1:1,000; guinea pig anti-SV2A (Synaptic Systems, 119 004) at 1:500; and rabbit anti-SV2B (Synaptic Systems, 119 102) at 1:500.

Techniques: Fluorescence, Labeling, Derivative Assay, Expressing, Inhibition, Transgenic Assay, Staining, Marker, In Situ, Liquid Chromatography with Mass Spectroscopy, Two Tailed Test, Whisker Assay, Transformation Assay, Western Blot, Control